Mycoplasma agassizii Multilocus Sequence Typing Using Nanopore Sequencing: Insights Into Genetic Diversity and Isolate Characterization

Mycoplasma agassizii (Mycoplasmopsis agassizii) is a major pathogen responsible for upper respiratory tract disease (URTD) in tortoises, contributing to worldwide population declines. Despite its significance, tools for strain-level identification and epidemiological tracking remain limited. This study proposes a Multilocus Sequence Typing (MLST) scheme based on eight housekeeping genes (uvrB, tpiA, gyrB, efTu, rpoB, uvrA, gmK, and atpG), combined with Nanopore sequencing, to characterize M. agassizii isolates from tortoises in Italy and Portugal. Among 127 samples, 73 (57.5%) tested positive for M. agassizii. Analysis of 15 isolates revealed 14 distinct sequence types (STs). High genetic diversity was observed, even among samples from the same rescue centers. No correlation was found between STs and geographic origin or tortoise species. Four loci (uvrB, gyrB, rpoB, and gmK) were sufficient to discriminate all STs at a haplotype diversity cut-off ≥ 0.83. The standardized index of association (Ia = 0.2246) suggests a partially clonal population with evidence of recombination. This MLST scheme offers a promising tool for tracking M. agassizii strain diversity and understanding its epidemiology. Broader application across symptomatic and asymptomatic hosts is needed to evaluate associations between specific strains and disease outcomes, which will support conservation and URTD control efforts in tortoises.